US6413750B1ExpiredUtility
(R)-specific alcohol dehydrogenases from lactobacillus with improved catalytic activity using an NAD+ substrate
Assignee: FORSCHUNGSZENTRUM JUELICH GMBHPriority: Mar 19, 1998Filed: Nov 18, 1999Granted: Jul 2, 2002
Est. expiryMar 19, 2018(expired)· nominal 20-yr term from priority
C12N 9/0006C12P 7/02
87
PatentIndex Score
90
Cited by
4
References
2
Claims
Abstract
In a method for improving the NADH specificity of preferred NADPH-dependent dehydrogenases, the alkalinity of the enzyme is reduced in the coenzyme docking area by an appropriate change of the amino acid sequence achieved by genetic engineering means. The inventive process is especially used for obtaining short-chain dehydrogenases with coenzyme binding areas on the N-terminus from Lactobacillus brevis and Lactobacillus kefir.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
# SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 27
<210> SEQ ID NO 1
<211> LENGTH: 31
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer BRS
<400> SEQUENCE: 1
gcgcgaattc atgtctaacc gtttggatgg t
#
# 31
<210> SEQ ID NO 2
<211> LENGTH: 52
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer BRAS
<400> SEQUENCE: 2
gcgcaagctt ctactattga gcagtgtagc caccgtcaac tacaaattca g
#a 52
<210> SEQ ID NO 3
<211> LENGTH: 36
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LK46I
<400> SEQUENCE: 3
accggcctgc acagcgatgt tggtgaaata gcagct
#
# 36
<210> SEQ ID NO 4
<211> LENGTH: 36
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LK46I rev
<400> SEQUENCE: 4
agctgctatt tcaccaacat cgctgtgcag gccggt
#
# 36
<210> SEQ ID NO 5
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer A9G
<400> SEQUENCE: 5
ggtaaggtag gaatcattac a
#
#
#21
<210> SEQ ID NO 6
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer A9G rev
<400> SEQUENCE: 6
tgtaatgatt cctaccttac c
#
#
#21
<210> SEQ ID NO 7
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LK45M
<400> SEQUENCE: 7
accggcctgc acagcgatgt tgaaatggca
#
# 30
<210> SEQ ID NO 8
<211> LENGTH: 33
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LK45M rev
<400> SEQUENCE: 8
tgccatttca ccaacatcgc tgtgcaggcc ggt
#
# 33
<210> SEQ ID NO 9
<211> LENGTH: 45
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LK48M
<400> SEQUENCE: 9
accggcctgc acagcgatgt tggtgaaaaa gcagctatga gtgtc
#
#45
<210> SEQ ID NO 10
<211> LENGTH: 45
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LK48M rev
<400> SEQUENCE: 10
gacactcata gctgcttttt caccaacatc gctgtgcagg ccggt
#
#45
<210> SEQ ID NO 11
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LH39L
<400> SEQUENCE: 11
accggcctgc tcagcgatgt t
#
#
#21
<210> SEQ ID NO 12
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: primer R38LH39L rev
<400> SEQUENCE: 12
aacatcgctg agcaggccgg t
#
#
#21
<210> SEQ ID NO 13
<211> LENGTH: 45
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: G37DR38LK48M
<400> SEQUENCE: 13
accgacctgc acagcgatgt tgttgaaaaa gcagctatga gtgtc
#
#45
<210> SEQ ID NO 14
<211> LENGTH: 45
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: G37DR38LK48M rev
<400> SEQUENCE: 14
gacactcata gctgcttttt caccaacatc gctgtgcagg tcggt
#
#45
<210> SEQ ID NO 15
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: wild-type
<400> SEQUENCE: 15
Ser Asn Arg Leu Asp Gly Lys Val Ala Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Gly Arg His Ser Asp Val G
#ly Glu Lys Ala Ala Lys
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 16
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: mutant 1
<400> SEQUENCE: 16
Ser Asn Arg Leu Asp Gly Lys Val Gly Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Gly Leu His Ser Asp Val G
#ly Glu Ile Ala Ala Lys
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 17
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: mutant 1/1
<400> SEQUENCE: 17
Ser Asn Arg Leu Asp Gly Lys Val Gly Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Gly Leu His Ser Asp Val G
#ly Glu Met Ala Ala Lys
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 18
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: mutant 2
<400> SEQUENCE: 18
Ser Asn Arg Leu Asp Gly Lys Val Gly Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Gly Leu His Ser Asp Val G
#ly Glu Lys Ala Ala Met
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 19
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: mutant 2/2
<400> SEQUENCE: 19
Ser Asn Arg Leu Asp Gly Lys Val Gly Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Gly Leu Leu Ser Asp Val G
#ly Glu Lys Ala Ala Met
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 20
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: mutant 2/3
<400> SEQUENCE: 20
Ser Asn Arg Leu Asp Gly Lys Val Gly Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Asp Leu His Ser Asp Val G
#ly Glu Lys Ala Ala Met
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 21
<211> LENGTH: 50
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: N-terminal area
<400> SEQUENCE: 21
Ser Asn Arg Leu Asp Gly Lys Val Ala Ile I
#le Thr Gly Gly Thr Leu
1 5
# 10
# 15
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
20
# 25
# 30
Val Met Ile Thr Gly Arg His Ser Asp Val G
#ly Glu Lys Ala Ala Lys
35
# 40
# 45
Ser Val
50
<210> SEQ ID NO 22
<211> LENGTH: 34
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: LBADH
<400> SEQUENCE: 22
Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe V
#al Glu Glu Gly Ala Lys
1 5
# 10
# 15
Val Met Ile Thr Gly Arg His Ser Asp Val G
#ly Glu Lys Ala Ala Lys
20
# 25
# 30
Ser Val
SEQ ID NO 23
<211> LENGTH: 34
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: sp-Q93761
<400> SEQUENCE: 23
Gly Leu Gly Lys Ala Ile Ala Thr Thr Phe A
#la His Leu Gly Ala Ser
1 5
# 10
# 15
Val Ala Ile Ala Ala Arg Arg Leu Asp Val L
#eu Glu Lys Thr Ala Asp
20
# 25
# 30
Glu Ile
<210> SEQ ID NO 24
<211> LENGTH: 35
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: sp-P50161
<400> SEQUENCE: 24
Gly Ile Gly Ala Ala Ile Ala Val Ala Leu G
#ly Glu Arg Gly Ala Lys
1 5
# 10
# 15
Val Val Val Asn Tyr Ala His Ser Arg Glu A
#la Ala Glu Lys Val Val
20
# 25
# 30
Glu Gln Ile
35
<210> SEQ ID NO 25
<211> LENGTH: 34
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: sp-O067610
<400> SEQUENCE: 25
Gly Ile Gly Arg Ala Ile Ala Glu Lys Leu A
#la Ser Ala Gly Ser Thr
1 5
# 10
# 15
Val Ile Ile Thr Gly Thr Ser Gly Glu Arg A
#la Lys Ala Val Ala Glu
20
# 25
# 30
Glu Ile
<210> SEQ ID NO 26
<211> LENGTH: 33
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: sp-P42317
<400> SEQUENCE: 26
Gly Ile Arg Phe Glu Ile Ala Arg Glu Phe A
#la Arg Glu Gly Ala Ser
1 5
# 10
# 15
Val Ile Val Asp Leu Arg Pro Glu Ala Cys G
#lu Lys Ala Ala Ser Lys
20
# 25
# 30
Leu
<210> SEQ ID NO 27
<211> LENGTH: 18
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus brevis
<220> FEATURE:
<223> OTHER INFORMATION: sp-Q49721
<400> SEQUENCE: 27
Glu Gly Leu Ile Gly Arg His Ala Asp Val G
#ly Ala Lys Val Ala Gln
1 5
# 10
# 15
Leu Ile
1. An (R)-specific alcohol dehydrogenase with a k cat /K m for NAD + of 20 to 66 mM −1 sec −1 ,
a) wherein said dehydrogenase is a mutant of a wild-type (R)-specific alcohol dehydrogenase described by the following characteristics:
(i) naturally-occurring from Lactobacillus,
(ii) having an N-terminal sequence as set forth in SEQ ID NO:15, and
(iii) using NADP −1 as a cofactor for catalytic activity;
b) wherein said mutant dehydrogenase has at least one mutation selected from the group consisting of: G37D, R38L, H39L, K45I, K45M, and K48M with respect to SEQ ID NO:15;
c) and wherein said mutation or mutations change the alkalinity of the cofactor binding site of the mutant dehydrogenase and promote catalytic activity using NAD + as a cofactor.
2. An (R)-specific alcohol dehydrogenase according to claim 1 , wherein said mutations are selected from the group consisting of:
a) R38L and K45I as shown in the N-terminal sequence set forth in SEQ ID NO:16,
b) R38L and K45M as shown in the N-terminal sequence set forth in SEQ ID NO:17,
c) R38L and K48M as shown in the N-terminal sequence set forth in SEQ ID NO:18,
d) R38L, H39L, and K48M as shown in the N-terminal sequence set forth in SEQ ID NO:19, and
e) G37D, R38L, and K48M as shown in the N-terminal sequence set forth in SEQ ID NO:20.Cited by (0)
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